Physiological immune surveillance is dependent on the continuous patrolling of lymphocytes between the blood and different lymphoid organs. In normal non-lymphoid tissue lymphocytes are absent or only present at a very low level, but in many inflammatory disease states vast numbers of lymphocytes can accumulate in various affected tissues and organs. One of the important molecules controlling lymphocyte exit from the blood is vascular adhesion protein-1 (VAP-1) disclosed in U.S. Pat. No. 5,580,780. VAP-1 is a homodimeric 170-180 kDa endothelial glycoprotein. VAP-1 mediates lymphocyte binding to venules in human tissue sections. VAP-1 is heavily glycosylated and the sugar moieties are important for the adhesion function (Salmi et al., 1996). Blocking the adhesive function of VAP-1 reduces the number of cells infiltrating inflamed tissue allowing the inflammation to resolve. VAP-1 is thus a target for anti-inflammatory drug development.
Human vascular adhesion protein-1 (VAP-1) is a membrane-bound multifunctional glycoprotein with both adhesive and enzymatic properties. The cloning of VAP-1 surprisingly revealed that it belongs to the semicarbazide-sensitive monoamine oxidases (SSAO; EC 1.4.3.6) (International Patent Publication WO 98/53049). VAP-1 is a type 2 integral membrane protein with a large catalytically active extracellular domain. Thus VAP-1 is an ectoenzyme. Neither the role of SSAO activity nor its physiological substrates in leukocyte-endothelial interaction is well defined. VAP-1 was the first molecularly defined transmembrane member of this enzyme group in mammals, and it accounts for 90% of cellular SSAO activity. Notably, SSAOs are different from the well-characterized monoamine oxidases A and B in respect to subcellular localization, substrates, cofactors, inhibitors, and protein sequence.
Although the SSAO reaction has been known since the 1950's in biochemical terms, the physiological function(s) of these enzymes has remained enigmatic. The physiological substrates of SSAO are not known. Two potential candidates, methylamine and aminoacetone, are however formed during intermediary metabolism in humans and can be de-aminated by SSAO in vitro and in vivo.
VAP-1 SSAO activity has been proposed to be directly involved in the pathway of leukocyte adhesion to endothelial cells by a novel mechanism involving direct interaction with an amine substrate presented on a VAP-1 ligand expressed on the surface of a leukocyte (Salmi et al., 2001). This publication describes the direct involvement of VAP-1 SSAO activity in the process of adhesion of leukocytes to endothelium. Thus inhibitors of VAP-1 SSAO activity could be expected to reduce leukocyte adhesion in areas of inflammation and thereby reduce leukocyte trafficking into the inflamed region and therefore the inflammatory process itself.
In human clinical tissue samples expression of VAP-1 is induced at sites of inflammation. This increased level of VAP-1 can lead to increased production of H2O2 generated from the action of the VAP-1 SSAO extracellular domain on monoamines present in the blood. This generation of H2O2 in the localised environment of the endothelial cell could initiate other cellular events. H2O2 is a known signalling molecule that can upregulate other adhesion molecules and this increased adhesion molecule expression may lead to enhanced leukocyte trafficking into areas in which VAP-1 is expressed. Other products of the VAP-1 SSAO reaction may also have biological effects also contributing to the inflammatory process. Thus the products of the VAP-1 SSAO activity may be involved in an escalation of the inflammatory process, which could be blocked by specific SSAO inhibitors.
VAP-1 SSAO may be involved in a number of other pathological conditions associated with an increased level of circulating amine substrates of VAP-1 SSAO. The oxidative deamination of these substrates would lead to an increase in the level of toxic aldehydes and oxygen radicals in the local environment of the endothelial cell which could damage the cells leading to vascular damage. Increased levels of methylamine and aminoacetone have been reported in patients with Type I and Type II diabetes and it has been proposed that the vasculopathies such as retinopathy, neuropathy and nephropathy seen in late stage diabetes could be treated with specific inhibitors of SSAO activity.
The development of specific VAP-1 SSAO inhibitors that modulate VAP-1 activity would be useful for the treatment of acute and chronic inflammatory conditions or diseases such as chronic arthritis, inflammatory bowel diseases, and skin dermatoses, as well as diseases related to carbohydrate metabolism (including diabetes and complications resulting from diabetes, such as vasculopathies). In addition, aberrations in adipocyte dfferentiation or function and smooth muscle cell function (in particular, atherosclerosis), and various vascular diseases may be suitable for treatment with VAP-1 SSAO inhibitors.
International Patent Publication WO 03/006003 discloses carbocyclic hydrazino compounds as well as the use thereof as inhibitors of semicarbazide-sensitive amine oxidases (SSAO), including human Vascular Adhesion Protein-1 (VAP-1).
Copper-containing amine oxidases (CAOs; EC 1.4.3.6) belong to the functionally diverse superfamily of amine oxidases (Dawkes et al., 2001). They are also known as semicarbazide-sensitive amine oxidases since their enzymatic activity can be blocked by a carbonyl-reactive compound, semicarbazide. They catalyse the oxidative deamination of primary amines to the corresponding aldehydes in a copper-dependent reaction where molecular oxygen is consumed and hydrogen peroxide and ammonia are released. A characteristic feature for all CAOs is the use of 2,4,5-trihydroxyphenylalanine quinone, a topaquinone (TPQ), as a redox cofactor. CAOs have been isolated from several different organisms, including bacteria, fungi, plants and mammals. In plants CAOs are involved, e.g. in wound healing, whereas in prokaryotes CAOs allow the organism to utilize various amines metabolically as sources of nitrogen and carbon. In higher eukaryotes very little is known about the biological function of CAOs besides their role in the metabolism of biogenic and other amines.
Shepard et al., 2002, report striking differences in selectivity and rates of inactivation when testing inhibitors against six known copper-containing amine oxidases.
The crystal structures of CAOs have been solved from four different species: Escherichia coli (ECAO; e.g. Protein Data Bank, PDB code 1oac) (Parsons et al., 1995), Pisum sativum (PSAO; PDB code 1ksi) (Kumar et al., 1996), Hansenula polymorpha (HPAO; e.g. PDB code 1a2v) (Li et al., 1998) and Arthobacter globiformis (AGAO; e.g. PDB code 1av4) (Wilce et al., 1997). All of these homodimeric structures have a similar overall fold that can be divided into domains D1-D4 of which the D1 domain is found only in E. coil. Domains D2 and D3 are ˜100 amino adds each and have an α/β type fold, whereas the largest, C-terminal domain D4 is ˜400 amino adds in length and has a unique β-sandwich fold that is needed for dimerization. The active site, which is located in the D4 domain, is highly conserved within the CAO family. It is buried deeply within the protein and accessable only via a long channel surrounded mainly by amino acids from the D3 and D4 domains. The amino acid residues of the D3 domain are less conserved than the actual active site, suggesting that the cavity leading to the active site is of great importance in determining the substrate specificity of CAOs.
Even though the structures of known CAO proteins are quite similar, the sequence identity at the amino acid level is only 25-35%. The evolutionary relationship of VAP-1 to the structurally known members of the CAO family has not been characterized, but the presence of a transmembrane domain at the N-terminus of VAP-1 suggests substantial divergence from the soluble CAOs.
The present invention provides the crystallization and X-ray analysis of human VAP-1. This is the first mammalian CAO to be crystallized.